The sequencing of entire genomes has resulted in the identification of large numbers of open reading frames (ORFs). Currently, significant effort is devoted to understanding gene function by mRNA expression patterns and by gene disruption phenotypes. Important advances in this effort have been possible, in part, by the ability to analyze thousands of gene sequences in a single experiment using gene chip technology. However, much information about gene function comes from the analysis of the biochemical activities of the encoded protein.
Currently, these types of analyses are performed by individual investigators studying a single protein at a time. This is a very time-consuming process since it can take years to purify and identify a protein based on its biochemical activity. The availability of an entire genome sequence makes it possible to perform biochemical assays on every protein encoded by the genome.
To this end, it would be useful to analyze hundreds or thousands of protein samples using a single protein chip. Such approaches lend themselves well to high throughput experiments in which large amounts of data can be generated and analyzed. Microtiter plates containing 96 or 384 wells have been known in the field for many years. However, the size (at least 12.8 cm×8.6 cm) of these plates makes them unsuitable for the large-scale analysis of proteins because the density of wells is not high enough.
As noted above, other types of arrays have been devised for use in DNA synthesis and hybridization reactions, e.g., as described in WO 89/10977. However, these arrays are unsuitable for protein analysis in discrete volumes because the arrays are constructed on flat surfaces which tend to become cross-contaminated between features.
Photolithographic techniques have been applied to making a variety of arrays, from oligonucleotide arrays on flat surfaces (Pease et al., 1994, “Light-generated oligonucleotide arrays for rapid DNA sequence analysis,” PNAS 91:5022-5026) to arrays of channels (U.S. Pat. No. 5,843,767) to arrays of wells connected by channels (Cohen et al., 1999, “A microchip-based enzyme assay for protein kinase A,” Anal Biochem. 273:89-97). Furthermore, microfabrication and microlithography techniques are well known in the semiconductor fabrication area. See, e.g., Moreau, Semiconductor Lithography: Principals Practices and Materials, Plenum Press, 1988.
Recently devised methods for expressing large numbers of proteins with potential utility for biochemical genomics in the budding yeast Saccharomyces cerevisiae have been developed. ORFs have been cloned into an expression vector that uses the GAL promoter and fuses the protein to a polyhistidine (e.g., HISX6) label. This method has thus far been used to prepare and confirm expression of about 2000 yeast protein fusions (Heyman et al., 1999, “Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation,” Genome Res. 9:383-392). Using a recombination strategy, about 85% of the yeast ORFs have been cloned in frame with a GST coding region in a vector that contains the CUP1 promoter (inducible by copper), thus producing GST fusion proteins (Martzen et al., 1999, “A biochemical genomics approach for identifying genes by the activity of their products,” Science 286:1153-1155). Martzen et al. used a pooling strategy to screen the collection of fusion proteins for several biochemical activities (e.g., phosphodiesterase and Appr-1-P-processing activities) and identified the relevant genes encoding these activities. However, strategies to analyze large numbers of individual protein samples have not been described.
Thus, the need exists for a protein chip in which the wells are densely packed on the chip so as to gain cost and time advantage over the prior art chips and methods.
Citation or identification of any reference in Section II or any other section of this application shall not be considered as admission that such reference is available as prior art to the present invention.